This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sequence-specific targeting of double stranded DNA (dsDNA) with exogenous nucleic acid probes remains an essentially unsolved challenge of biological chemistry. This is unfortunate since a general dsDNA targeting methodology potentially would yield highly rewarding outcomes including development of: powerful diagnostic probes for fluorescence in situ hybridization (FISH) studies, research tools for gene modulation, and potential drug candidates that may prevent formation of a disease-related protein at a very early stage (improved antigene strategy). The proposed project seeks to further refine and throughly characterize a recently discovered dsDNA targeting methodology, which is based on intercalator-modified nucleic acids. More specifically, DNA duplex probes with +1 interstrand arrangements of pyrene-functionalized 2'-amino-[unreadable]-L-LNA (Locked Nucleic Acid) monomers are very unstable (intercalator-induced duplex unwinding), while each of the strands exhibit an enormous affinity toward complementary DNA as a consequence of precise positioning of the intercalator in the duplex core. This thermal advantage is exploited as a driving force to facilitate fast sequence-specific recognition of dsDNA targets at physiologically relevant salt concentrations, which conveniently can be followed by a fluorescence assay. Herein, we propose to optimize this molecular recognition process by fine-tuning the chemical building blocks via attachment of more efficient intercalators (e.g., perylene, coronene or ethidium bromide analogs) and/or by synthesizing the more convenient O2'-alkylated RNA or N2'-functionalized 2'-amino DNA analogs of these building blocks. Further we propose to carry our an extensive series of biophysical characterization experiments (thermal denaturation, fluorescence, strand exchange, NMR, molecular modeling, etc ) to utilize this information for rational design of novel more efficient analogs, and to evaluate the full diagnostic/therapeutic potential of this methodology.